The overall purpose of this NIH Pathway to Independence Award is for Dr. Hall to gain the additional training needed to develop into an independent investigator capable of conducting studies to investigate interactions between nutritional and environmental factors. Building upon a background in nutrition and epidemiology, the mentored phase of this award will provide training in 1) study management and oversight, 2) advanced nutritional biochemistry and the mathematical modeling of one-carbon metabolism, and 3) laboratory sciences. The acquired skills will be applied to the current area of research, investigating the influence of nutrients involved in one-carbon metabolism on the methylation of both arsenic (As) and genomic DNA. Specifically, the roles of two nutrients, choline and betaine, i.e. key nutrients involved in methylation pathways, which have not previously been considered in relation to As and DNA methylation, will be examined. Roughly 140 million people in over 70 countries are chronically exposed to As-contaminated drinking water at concentrations far exceeding the World Health Organization standard of 10 5g/L. As is a class I carcinogen known to cause cancers of the skin, bladder, and lung, as well as ischemic heart disease and neurologic impairments. Methylation of ingested inorganic arsenic (InAs) to methylarsonic- (MMA) and dimethylarsinic acids (DMA) relies on nutrient-dependent one carbon metabolism and facilitates urinary As elimination. Methylation of DNA via one-carbon metabolism is an epigenetic modification that plays critical roles in the regulation of gene expression and maintenance of chromosomal stability, and may play a role in the underlying mechanism of As-induced carcinogenesis. In the mentored phase, the influence of choline and betaine on methylation of As and DNA will be investigated in an existing cross-sectional study of 375 Bangladeshi adults exposed to a wide range of As concentrations in drinking water. In the independent phase, the skills acquired during the training will be applied to further investigate the roles of choline and betaine, including their interactions with other nutrients, i.e. folate and creatine, in the methylation of both As and DNA utilizing data and biospecimens from a randomized controlled trial of 600 individuals chronically exposed to As. In addition, mathematical models of one-carbon metabolism will be employed to conduct in silico experiments in conjunction with the clinical trial. These experiments will 1) allow the models to be further validated against the clinical trial data and 2) provide a deeper mechanistic understanding of the results from the trial. At the conclusion of this award, Dr. Hall will have developed into an independent multidisciplinary investigator well positioned to develop additional hypotheses related to the role of nutrient/environment interactions in disease development. Public Health Relevance: On a global basis, WHO estimates that more than 140 million people in at least 70 countries are exposed to arsenic (As)-contaminated drinking water, most in the developing world where nutritional deficiencies are common. The proposed studies of choline and betaine build considerably upon the goals of the research of the parent studies, which is to identify interventions to reduce blood As concentrations (i.e. internal As exposure) through simple, low-cost, low-risk nutritional manipulations of one- carbon metabolism. Previous work by our group has shown that folic acid supplementation in folate-deficient As-exposed individuals facilitated As elimination and significantly lowered blood As concentrations. However, the finding that there were also many individuals who did not appear to be responsive to folic acid suggests the possibility that there is room to improve on the efficacy of FA and that some individuals could potentially benefit from another therapeutic modality such as choline or betaine. Considering the magnitude of the exposed population worldwide, and the severity of the numerous associated health outcomes, the investigators feel this work is highly significant as it implies that simple, low-cost, low-risk interventions could have therapeutic potential for ameliorating the long-term health consequences for the many populations at risk.